I have been fixing epithelial cells (A-549s) with either 4% or 3.2% PFA for sometime. Even when the PFA is fresh and frozen into aliquots, the cells end up looking very ragged and leaky. Most of them also end up peeling up and sloughing off during washes, leaving only clumped/sticky cells... and very few of those to boot.

Anybody have any ideas? We've tried several different PFA lots, so I don't think the PFA we are getting is bad.

Thanks,Chris

-Ghengis Corn-

Could you switch to acetone fixation? It is very quick, but it doesn't work for all proteins.d

-rosewater-

Yeah, I also might try methanol fixation, or even several lower concs. of PFA (like 0.5%, 1%, etc...). I'd need to be able to maintain proteins-bound-to-proteins on the cell surface. For that I don't think I need to worry about epitopes getting changed...